Abstract In this application, we will test a longstanding but effectively untested hypothesis in myelin biology, that white matter recovery after sustained or recurrent demyelination might be constrained by the finite mitotic competence of the human glial progenitor cell pool. Such a mobilization-dependent depletion of mitotically- competent progenitors might lead to mitotic senescence, and hence to the eventual failure of remyelination noted in progressive multiple sclerosis. In addition, any such depletion of competent progenitor cells might also be expected to limit the utility of differentiation-based approaches towards induced remyelination. We thus propose to assess the responses of human glial progenitor cells (hGPCs) to demyelination in vivo, by defining the single cell RNA expression patterns of hGPCs, both at baseline and in response to sustained cuprizone demyelination in vivo. To this end, we will use mice neonatally chimerized with human GPCs, a novel model we have developed in which mouse oligodendrocytes and astrocytes are largely replaced by their human counterparts in vivo. Using these human glial chimeras, we will ask the following questions: 1) What is the phenotypic and transcriptional heterogeneity among single human glial progenitor cells in vivo, in the otherwise undisturbed adult glial chimeric brain? Are all GPCs multi-lineage competent? Are some more restricted than others to astrocytic or oligodendrocytic fate? Are some in cell cycle while others are more quiescent? How do these phenotypic distributions change with age? 2) How heterogeneous are the transcriptional responses of resident human GPCs to demyelination in vivo? In response to cuprizone-mediated demyelination, what differentially regulated pathways distinguish quiescent, initially mobilized, and actively remyelinating hGPCs? These experiments will combine the use of human glial chimeras engrafted with genetically tagged GFP+ hGPCs, with later single cell RNA-seq of both the post-demyelination white matter, and of pooled hGPC isolates after post-demyelination FACS, to define the transcriptional events associated with human GPC mobilization and remyelination in vivo. 3) Does the efficiency of remyelination by hGPCs fall with sustained demyelination? Are human GPCs capable of self-renewal during sustained demyelination, or is remyelination delimited by their mitotic senescence? These experiments will assess both methylation state and telomeric length of GPCs in vivo, both before and after sustained cuprizone exposure, so as to define the effects of sustained demyelination on these hallmarks of cellular aging. In addition, we will assess the transcriptional concomitants to methylation state-defined aging, by RNA-seq of the same cells as a function of time after demyelination. By this means, we intend to define both the transcriptional hallmarks of mitotic exhaustion by hGPCs, and the epigenetic correlates to that process, and by doing so to identify therapeutic targets by which to delay or control the demyelination-associated depletion of mitotically-competent hGPCs.